This disclosure relates to inhibition of STAT3 activity.
AMD is a rapidly growing retinal disease which primarily affects patients of age 50 years and older. Current prevalence rates in the US estimate that over 15 million US citizens are afflicted with this disorder; however, as a consequence of the rapidly growing aging population, it is predicted that the number of persons afflicted with AMD will increase 50% by 2020. Patients with AMD present with loss of their central vision that progressively worsens with age. There are two major classifications of AMD, dry and wet (or non-exudative and exudative). All patients will initially present with Dry-AMD, and approximately 85-90% of all AMD patients have the dry form of the disease. Dry-AMD features a progressive degeneration in the retinal pigment epithelial (RPE) cells, Bruch's membrane, and choroid. Another characteristic phenotypic feature is the development of subretinal deposits called drusen. These drusen have been studied with proteomics and are a compilation of numerous proteins and toxic molecules. Importantly, drusen also contain numerous inflammatory molecules that are known to initiate inflammatory cascades. Therefore, current hypotheses regarding Dry-AMD pathogenesis revolve around uncontrolled inflammation in the RPE. Since this disease primarily affects elderly patients, it is thought that years of oxidative stress lead to the initial formation of drusen, which causes the body to elicit an inflammatory response to these drusen, which then exacerbates further drusen formation. As the RPE is required to support the function of the retinal photoreceptors, this insult causes degeneration of the photoreceptors which leads to vision loss. Moreover, this inflammation leads to a breakdown of the blood retinal barrier, and formation of choroidal neovascularization (CNV) that occurs underneath the macula which is characteristic of Wet-AMD. This abnormal neovascularization causes vascular leakage of blood and fluid into the retina which leads to further visual loss in Wet-AMD patients. The progressive vascular leakage then exacerbates retinal and RPE insult and causes accelerated photoreceptor apoptosis and initiation of inflammatory pathways, which can permanently inhibit the potential for therapeutic intervention.
Several studies have demonstrated that inflammation plays a crucial role in the pathogenesis of retinal diseases and AMD. These patients have several characteristics of chronic inflammatory diseases, such as increased nitric oxide production, intracellular adhesion molecule-1 (ICAM-1) up-regulation, leukostasis and increased vascular permeability. It has been shown that the patients with proliferative retinopathies have elevated serum pro-inflammatory markers, such as TNF-α, C-reactive peptide (CRP), interleukin-1 (IL-1), IL-6, IL-18, soluble ICAM-1, and circulating vascular cell adhesion molecule-1 (VCAM-1). Induction of MCP-1, IL-8 and TNF-α is also implicated in ischemia-induced retinal NV. Recent evidence has also shown that VEGF induces ICAM-1 expression and leukostasis in the retina, suggesting that VEGF is a pro-inflammatory factor.
Several large genetic population studies have been carried out to identify susceptibility loci contributing to AMD development. These studies have identified over 30 loci demonstrating significant correlation to the risk of developing AMD, depending on the population studied. Based on these results, it is suggested that AMD is a complex disease that occurs as a result of several environmental and genetic disposition elements; however, these association studies have identified a number of inflammatory-related genes that predispose individuals to AMD. In multiple populations, mutations in the complement factor H (CFH) gene have been identified that are present in a significant number of AMD afflicted individuals. CFH is present in the bloodstream and is a inhibitor of complement activation. Thus, it has been suggested that the CFH mutations observed in AMD patients may represent an inability for these individuals to restrict inflammation in the retina as a result of vascular leakage. In addition, another large association study has implicated variants of toll-like receptor 4 (TLR4) in contributing to AMD susceptibility. TLR4 has been demonstrated to play a significant role in pro-inflammatory signaling pathways, which may also contribute to the inflammation observed in AMD pathogenesis. Another TLR family member, TLR3, has been recently implicated in Dry-AMD formation. This large genetic study demonstrated that an allele of TLR3 was associated with Dry-AMD development degeneration of RPE cells. Moreover, the authors identified a protective TLR3 allele that was associated with prevention of Dry-AMD development and progression. This protective allele reduced the ability of TLR3 to activate inflammatory pathways that eventually result in RPE cell apoptosis.
It has been shown that multiple growth factors, such as VEGF, bFGF, IGF-1, PEDF, etc. in the eye are implicated in the pathogenesis of retinal diseases and AMD. Alterations of these growth factors and their receptors in diabetes have been identified in both experimental and clinical studies. VEGF is a potent mediator of vascular permeability leading to angiogenesis and a potent mitogen with a unique specificity for endothelial cells in a variety of human pathological situations. The increased VEGF levels are responsible for the retinal vascular leakage or retinal vascular hyper-permeability, and retinal neovascularization. A number of clinical and animal studies have shown that VEGF plays a pivotal role in the development of AMD. The increased expression of retinal VEGF and its receptors correlate to high retinal vascular permeability in animal models of retinal disease with neovascularization. Inhibition of VEGF and VEGF receptors can prevent retinal NV in animal models. The causative role of VEGF in AMD pathogenesis has also been well established by many animal and clinical studies. Current pharmacological treatments for AMD are inhibitors that bind VEGF and prevent subsequent initiation of pathways leading to neovascularization. Although these compounds have has success in the clinic, they fail to address the inflammatory nature of the disease.
Leptin is an adipocyte-derived cytokine that has been linked to obesity in both humans and other animal models. Activation of the leptin receptor (Lep-R), a member of the gp130 receptor family, triggers a cascade of phosphorylation events that lead to changes in cellular gene expression. Clinical studies have demonstrated that patients with diabetic retinopathy, amongst other proliferative retinal disorders, have significantly increased levels of leptin in their vitreous humor. Leptin stimulation has been shown to exert a pro-angiogenic effect both in vitro and in vivo. Treatment of human vascular endothelial cells with leptin causes a rapid phosphorylation of the transcription factor STAT3 leading to angiogenesis.
STAT3 is a transcription factor that exerts a positive effect to promote expression of several angiogenic growth factors, such as VEGF and platelet derived growth factor, and has been shown to be constitutively active in several tumors and transformed cell types. Upon phosphorylation at tyrosine 705, STAT3 monomers dimerize and translocate to the nucleus to exert an effect on gene expression. The activated pSTAT3 is known to positively regulate VEGF through a STAT3-binding site on the VEGF promoter. Additionally, pSTAT3 also causes an upregulation of several proinflammatory molecules.
The pro-angiogenic effect of leptin stimulation is mediated by the upregulation of VEGF and can be inhibited by expression of a dominant-negative STAT3 variant. Furthermore, in the OIR mouse model, mice overexpressing leptin develop more severe retinal NV, while those deficient for leptin showed markedly suppressed retinal NV. A recent study has also demonstrated that activation of STAT3 by IL-6 stimulation can result in choroidal NV, and blockade of this pathway could inhibit NV formation. STAT3 expression is observed in both the inner nuclear layer and inner plexiform layer of the retina; however, pSTAT3 is localized exclusively in neovascular retinal vessels, which suggests an intimate involvement of pSTAT3 in the formation of retinal NV.
IL-6, an inflammatory cytokine, has also been linked to the progression of Dry-AMD to Wet-AMD. A clinical study published in 2005 enrolled patients garnering early stage characteristics of Dry-AMD. The authors quantified systemic levels of IL-6 at study enrollment and at a follow-up date approximately 4 years later. The authors demonstrated a direct correlation between increased IL-6 levels and progression from dry to Wet-AMD, potentially implicating IL-6 in AMD pathogenesis and establishing it as a biomarker for disease progression. Other studies using animal models have also demonstrated that IL-6 is directly implicated in retinal neovascularization, and prevention of IL-6 signaling could attenuate neovascularization. Specifically, the authors demonstrated that inhibition of IL-6 prevented activation of STAT3 in this model, which was the mechanism to attenuate neovascularization
The role of reactive oxygen species (ROS) and oxidative stress have been well established in the development of AMD-like phenotypes in several animal models. In vascular cells, a major source of ROS arises from the activity of NADPH oxidase. NADPH oxidase is a critical mediator in the downstream development of ischemia induced VEGF expression that leads to angiogenesis. Furthermore, the inhibition of NADPH oxidase prevents early inflammatory events, such as leukostasis, the lead to breakdown of the blood retinal barrier and subsequent angiogenesis. Recently, it was demonstrated that STAT3 is responsible for promoting NADPH oxidase overexpression in the retina, and inhibition of this pathway could prevent retinal inflammation, neovascularization and breakdown of the blood retinal barrier. Another recent paper demonstrated that elevated STAT3 activation during retinal inflammation leads to the ubiquitin-proteasome dependent degradation of the master phototransduction molecule, Rhodopsin. As this protein is central to the process of phototransduction, any deficiencies in Rhodopsin levels can lead to retinal degeneration and loss of visual function.
There is currently no FDA-approved therapeutics or procedures to treat Dry-AMD. The only major clinical study of significance is the Age-Related Eye Disease Study (AREDS) that was conducted by the National Eye Institute (NEI) and concluded in 2001. The study assessed the use of orally-administered antioxidants and zinc to prevent progression of Dry-AMD. The study demonstrated that high-levels of antioxidants and zinc could reduce risk of vision loss in patients with late stages of Dry-AMD; however, the treatment had no effect on patients with early or intermediate stages of the disease, for example, patients presenting with drusen that currently have little or no vision loss. In addition, the benefit of this antioxidant/zinc regimen appears to only be effective in certain populations of affected individuals. Thus, there is still a great demand to develop Dry-AMD therapies that can prevent vision loss altogether. As demonstrated by ongoing clinical trials of experimental therapeutics, the FDA-accepted endpoint for Dry-AMD treatment is the quantification of drusen number and volume, in addition to visual acuity measurements.